Abnormalities in the expression of genes, both in the timing and level of expression of particular genes, are a fundamental cause of cancer and other human disease. Abnormalities in genomic DNA, i.e. in chromosomes, are also a fundamental cause of cancer and other human disease, often leading to the over-expression or under-expression of genes. Some chromosomal abnormalities, such as balanced translocations and inversions between chromosomes, and base pair changes, do not involve a change in DNA sequence copy number. Other genomic DNA abnormalities comprise changes in DNA sequence copy number from the normal two copies per cell. These genomic DNA abnormalities often are referred to as gene amplification for copy number increase and gene deletion for copy number decrease. For example, one aggressive form of breast cancer, occurring in about 25-30% of breast cancers, results from the gene amplification and over-expression of the Her-2/neu oncogene, which is located on chromosome 17 at band q12. Breast cancer patients with this genetic abnormality have a significantly poorer prognosis, both for overall survival and disease-free survival, then patients without this abnormality. Proper assessment and management of breast cancer thus requires tests to measure the presence of Her-2 gene chromosomal copy number.
Chromosomal abnormalities such as Her-2 gene copy number can be assessed by assays using fluorescent in situ hybridization (“FISH”). FISH assays involve hybridization of DNA probes to chromosomal DNA present in morphologically intact metaphase spreads or interphase cells of tissue samples. The U.S. Food and Drug Administration recently approved a diagnostic FISH test, PathVysion™ HER-2, available from Vysis, Inc. (Downers Grove, Ill.) for detection of HER-2 copy number and prediction of outcome of anthracyclin therapy in node positive breast cancer patients.
Cancer also involves abnormalities in multiple genes, leading to multiple forms of the disease, as exemplified by breast cancer, wherein the Her-2 oncogene is not abnormal in the majority of cases. So-called “DNA Chip” or “microarray” tests using hybridization to a two dimensional array of multiple nucleic acid probes attached to a solid substrate assess multiple gene expression abnormalities simultaneously. See for example, U.S. Pat. No. 5,445,934, “Array of Oligonucleotides on Solid Substrate,” Fodor, et al., U.S. Pat. No. 5,800,992, “Method of Detecting Nucleic Acids,” Fodor, et al., and U.S. Pat. No. 5,807,552, “Methods for Fabricating Microarrays of Biological Substances,” Brown, et al. The microarray gene expression tests are of growing use in the development of new drugs targeted at particular diseases.
Multiple gene expression at the protein level also can be examined by the use of “microdot” immunoassays, which are two dimensional arrays of immobilized antigens on a substrate. See U.S. Pat. No. 5,486,452, “Devices and Kits for Immunological Analysis,” Gordon, et al., priority date Feb. 3, 1982, and Ekins, et al, Analytica Chimica Acta, 227:73-96 (1989). The immobilized antigens of Gordon, et al. include nucleic acids and are disclosed as arrayed at densities of 105 per 10 square centimeters (or 1,000 per cm2). Gordon, et al. further disclose the array has “intrinsic resolution” below the size of pipetting devices common in 1982, see Gordon, et al. at column 17, and can thus contain antigens at higher densities. Gordon, et al. disclose that the arrays can be manufactured by use of mechanical transfer apparatus, miniaturized applicators, lithographic procedures or high speed electronic printing.
U.S. Pat. No. 5,665,549, “Comparative Genomic Hybridization (CGH),” Pinkel, et al., discloses a method for simultaneous assessment of multiple genetic abnormalities. CGH involves the comparative, multi-color hybridization of a reference nucleic acid population labeled in one fluorescent color and a sample nucleic acid population labeled in a second fluorescent color to all or part of a reference genome, such as a human metaphase chromosome spread. Comparison of the resulting fluorescence intensity at locations in the reference genome permits determination of copy number of chromosomal sequences, or of expressed gene sequences, in the sample population. Microarray-based CGH tests have also been disclosed for the assessment of multiple genomic DNA or gene expression abnormalities, see U.S. Pat. No. 5,830,645, “Comparative Fluorescent Hybridization to Nucleic Acid Arrays, Pinkel, et al.; co-pending and commonly assigned U.S. patent application Ser. No. 09/085,625, “Improvements of Biological Assays for Analyte Detection,” Muller, et al.; and Pinkel, et al., “High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays,” Nature Genetics, Vol. 20, October 1998, pp. 207-211. Pinkel, et al. in Nature Genetics disclose the capability of CGH to a microarray target to detect a single copy change in genomic DNA.
The PathVysion HER-2 FISH assay was the first in vitro diagnostic test approved by the FDA for clinical use to predict cancer therapeutic outcome based upon a nucleic acid probe test of patient tissue. In its approved clinical use, the PathVysion assay allows the stratification of node positive breast cancer patients into two groups: (i) HER-2 amplified and likely to respond to high dose anthracyclin therapy and (ii) HER-2 non-amplified and unlikely to need high dose anthracyclin therapy. This classification is highly significant because use of high dose anthracyclin therapy entails a higher risk of cardiotoxicity than lower dosage. What is needed for clinical cancer management are additional in vitro diagnostic tests such as the PathVysion assay that will allow the clinician to further refine cancer therapy selection for a particular patient.
It is an object of this invention to stratify cancer patients into various cancer therapy groups based on analysis of disease tissue using a genomic DNA microarray for multiple gene amplifications or deletions present or absent in the diseased tissue of the patient. It is another object to stratify a particular patient into one of at least four different cancer therapy groups based on the microarray analysis of gene amplification or gene deletion at multiple chromosome locations. It is a further object to stratify a particular patient into one of at least nine different cancer therapy groups based on the microarray analysis. Other objects of the invention will be detailed below.